Project Summary ? Project 2 Melanoma, the most aggressive form of skin cancer, is characterized by a very high rate of somatic mutations. Approximately 50% of cutaneous melanomas have a mutation that affects the V600 residue in Braf. The activity of the mutant form of the protein (BRAFV600) is markedly increased and results in constitutive activation of the RAS-RAF-MAPK signaling pathway. This information has rapidly translated into clinical benefit, as five targeted therapy regimens have been approved for the treatment of metastatic melanoma patients with a BrafV600 mutation. While these agents have very high clinical response rates, unfortunately the majority of patients develop resistance within 2 years. Further, these targeted therapies cannot be used in patients who do not have a BrafV600 mutation. Thus, there remain unmet clinical needs to identify strategies to prevent resistance to approved targeted therapies in metastatic melanoma patients with an activating BrafV600 mutation, and new targeted therapy approaches for patients without a BrafV600 mutation (BRAF Wild-Type). While many studies are ongoing to evaluate the therapeutic potential of other kinase inhibitors, there is growing evidence to support the rationale for the testing of agents that target the apoptotic machinery. In contrast to many other cancers, cutaneous melanomas have a low rate (~20%) of mutations in Tp53. Additional studies have shown anti-apoptotic members of the BCL2 family of proteins can promote resistance to targeted therapies against the RAS-RAF-MAPK pathway, and that pro-apoptotic proteins (i.e., BIM) are critical to their effectiveness. Based on promising results in a limited number of preclinical models, clinical trials targeting the apoptotic machinery in combination with RAS-RAF-MAPK pathway inhibitors are ongoing in metastatic melanoma patients. However, currently there are no biomarkers to optimize patient selection for these strategies, nor understanding of resistance mechanisms to them. The central hypothesis of this proposal is that specific molecular features will predict sensitivity and resistance to combinatorial strategies utilizing pro- apoptotic agents and MAPK pathway targeted therapies. In order to test this hypothesis we will evaluate pro-apoptotic agents in combination with MAPK pathway inhibitors in molecularly characterized melanoma PDX models, which accurately replicate the molecular features and heterogeneity of this disease. In AIM 1 we will evaluate the efficacy and molecular effects of navitoclax, a BH3 mimetic that inhibits BCL2, alone and in combination with dabrafenib (BRAFi) and trametinib (MEKi) in melanoma PDX with a BrafV600 mutation. These experiments mirror an ongoing randomized phase II of these agents in metastatic melanoma patients, and the results of that trial will be used to clinically validate markers associated with resistance in the PDX. In AIM 2 we will evaluate the efficacy of the MDM2 inhibitor AMG232, alone and in combination with trametinib. Testing will be performed in BRAF WT melanomas with wild-type Tp53, including a subset with MDM2 amplification. These studies will help to refine and prioritize strategies using these agents in metastatic melanoma patients.